Segment,Title,Date,Author,Text,Image,Thumb
PR,Let's start!,04/06/2015,Silvia,"First day in the lab! After months and months of brainstorming we are so excited to start working! We extracted the Proteorhodopsin (BBa_K773002) from the 2015 Registry Kit, plate 2 well 1P. We transformed competent cells and plated them. This membrane protein is one of the main genes of our project. Our idea was to add a constitutive promoter and RBS in order to create a functional translational unit. ",,
CYT,Let’s get it started,04/06/2015,Elisa,"The parts K917014 (pLac promoter + S. oneidensis tetraheme cytochrome c + cymA) and K1316012 (T7 lacO + mtrCAB) were extracted from the Registry of Standard Biological Parts, transformed into NEB10β cells and amplified. ",,
PNCB,Extraction of gene pncB,05/06/2015,Riccardo,We did a PCR with specific designed primers to extract the gene pncB from the genome of Escherichia Coli. We tried using the Phusion polymerase and both HF and GC buffers. We obtained a better result with buffer GC and the length of pncB is right about 1200 bp.,http://2015.igem.org/wiki/images/9/90/Unitn_pics_notebook_pncB_PCR_extraction.jpg,http://2015.igem.org/wiki/images/9/90/Unitn_pics_notebook_pncB_PCR_extraction.jpg
PR,Is PR correct?,06/06/2015,Silvia,The screening digestion confirmed the gene!,,
PNCB,Preparing the insertion of pncB in pSB1C3,09/06/2015,Riccardo,We digested the plasmid pSB1C3 and the PCR product of pncB with the restriction enzymes Spe1 and Xba1 and incubated at 37°C for 3h. We added 1 μL of CIP (alkaline phosphatase) in the plasmid pSB1C3 and 1 μL of Dpn1 in pncB.,,
CYT,"Checking the right parts

",09/06/2015,Elisa,"Today we purified the DNA, quantified it and then performed a screening digestion. Bad results, the two parts seems inverted.",,
PNCB,Ligation and transformation of pncB in psb1C3,10/06/2015,Riccardo,We prepared the ligation reaction between pSB1C3 and pncB. We transformed 10 μL of ligation product into competent cellular using the “trasformation protocol”.,http://2015.igem.org/wiki/images/9/90/Unitn_pics_notebook_reaction_of_ligation_1.jpg.png,http://2015.igem.org/wiki/images/d/d3/Unitn_pics_notebook_reaction_of_ligation_1.jpg_thumb.jpg
PR,The idea,11/06/2015,Silvia,"We want to perform a 3A assembly between PR (that is in pSB1C3), pSB1K3, and the promoter J23100 with RBS (BBa_K081005, that is in pSB1A2). We extracted from the registry the following parts: pSB1K3 form the 2015 Kit, plate 4 well 6B, and the promoter from 2012 Kit, plate 2 well 10F. We transformed competent cells with the DNA. Unfortunatelly it took us longer than expected because the enzymes did not work properly. ",,
CYT,Trying again,11/06/2015,Elisa,"To verify if we inverted the parts during the digestion, we took two other colonies from the plate and started over.",,
PNCB,Insertion of pncB in pSB1C3,12/06/2015,Riccardo,"We digested 5 miniprepped DNA samples with PstI and run them on a gel. Since pcnC contains an illigal site for PstI, if the insertion of the gene happened correctly we will see two bands on the gel (the plasmid + a portion of pncB); if it didn't we will just see one band corresponfing to the empty plasmid.
",http://2015.igem.org/wiki/images/9/92/Unitn_pics_notebook_pncBpsb1C3.jpg,http://2015.igem.org/wiki/images/9/92/Unitn_pics_notebook_pncBpsb1C3.jpg
CYT,Total frenzy!,13/06/2015,Elisa,"The result of the screening digestion was completely insane!
We suspected the presence of internal restrictions sites.",,
CYT,Starting again from the beginning,14/06/2015,Elisa,We decided to re-transform cells with the two parts.,,
CYT,"OK, there's a problem…",16/06/2015,Elisa,"Once again, the result of the screening digestion was totally crazy.
That time we didn’t know what else to do, so we decided to sequence the parts.",,
INTERLAB,The Beginning of A New Adventure,18/06/2015,Claudio,We enrolled in the Measurement InterLab Study.,,
PNCB,Elimination of the illegal site of Pst1,18/06/2015,Riccardo,We used specifically designed primers and Phusion PCR with buffer GC (100 pg/μL). This is the gel of the PCR of site directed mutagenesis. The one band on lane 2 is ~3300 bp.,http://2015.igem.org/wiki/images/f/ff/Unitn_pics_notebook_Gel_site_mutagenesis.jpg,http://2015.igem.org/wiki/images/f/ff/Unitn_pics_notebook_Gel_site_mutagenesis.jpg
INTERLAB,Extraction of InterLab parts,19/06/2015,Elisa,"The parts BBa_K823005, BBa_K823008, BBa_K823013 and BBa_I13504 were extracted from the Registry of Standard Biological Parts, transformed into NEB10β cells and amplified. ",,
CYT,…and now we know why!,20/06/2015,Elisa,Sequencing showed that the parts from the Registry are incorrect.,,
INTERLAB,Will those parts be the right ones?,21/06/2015,Claudio,"Each extracted part was screened by restriction digestion or PCR. More specifically, BBa_K823005, BBa_K823008, BBa_K823013 were confirmed by PCR using Phusion Polymerase. BBa_I13504 was confirmed by restriction digestion. ",,
PNCB,Habemus mutagenesis,21/06/2015,Riccardo,"It took three days to do the ligation of the PCR mutagenesis, the transformation, the O/N and the purification. We digested the five sample with PstI and run them on gel. All of them were mutated for the illegal site! ",http://2015.igem.org/wiki/images/5/5d/Unitn_pics_notebook_Gel_habemus_mutagenesi.jpg,http://2015.igem.org/wiki/images/5/5d/Unitn_pics_notebook_Gel_habemus_mutagenesi.jpg
PR,Will the enzymes finally cut?,22/06/2015,Silvia,"After some misadventures we could finally perform the 3A assembly. We digest the parts with the correct enzymes: pSB1K3 cut with E and P, J23100 with RBS with E and S, the Proteorhodopsin with X and P. After 3 hours of digestion and purification from the enzymes, the parts were ligated together, then competent cells were transformed with the ligation and plated. The parts had three different antibiotic resistances so we were able to screen them. In fact, the plates we used were supplemented with kanamicine: in this way the colonies grown have the Kan resistance and hopefully pSB1K3 with our inserts! ",,
INTERLAB,Building our measurement devices,23/06/2015,Elisa,"Today we performed a standard assembly to get our measurement devices. We used BBa_K823005, BBa_K823008, BBa_K823013 (the BioBricks containing the promoter sequence) as vectors, opening them with SpeI and PstI and inserting the GFP gene (BBa_I13504) by cutting it with XbaI and PstI.",,
PNCB,Ligation of mutated pncB in the plasmid with J23100 promoter,23/06/2015,Riccardo,It failed...,,
INTERLAB,Today’s “almost positive” results,25/06/2015,Claudio,Each newly device was screened by restriction digestion and by observing the presence of fluorescence on a trans-illuminator.  We also performed Colony PCR in order to screen the transformed cell lysate. Unfortunately the assembly did not happen., ,
PR,3A,25/06/2015,Silvia,"We miniprepped many colonies taken from the 3A and performed a screening digestion. The plasmids were cut with E and P, in this way we expected to see two bands: the plasmid backbone (2070 bp) and the promoter with RBS plus the proteorhodpsin, which should be around 835 bp. From the gel we had seen see that the colonies B - D - E may be correct: the plasmid band is evident, but it is not so easy to discriminate the dimension of the other band that is between 750 and 1000. In fact the PR is 777 bp, while the promoter with RBS is long 58 bp. For this reason we tried another gel with the PR only and Assembly but unfortunately the bands look similar. The only solution seems to sequence the DNA!",http://2015.igem.org/wiki/images/3/3a/Unitn_pics_notebook_notebook_3A.jpg,
CYT,Never give up!,25/06/2015,Elisa,"Despite the bad news we won’t give up! We decided to contact Dr. M. Caroline Ajo-Franklin from C. Ajo-Franklin Lab, Berkeley.",,
INTERLAB,"Trying again, will this be the right time?",26/06/2015,Elisa,"We set again the assembly, this time with an O/N digestion. BBa_I13504 was amplified by PCR with Phusion polymerase, purified, and digested with XbaI and PstI overnight at 37°C. BBa_K823005, BBa_K823008, BBa_K823013 were digested with SpeI and PstI. The following day 1μL of SAP phosphatase was added to the destination vector (i.e. the one containing the promoter), and 1μl of DpnI was added to the GFP digestion product. Both samples were incubated at 37°C for 2 hours. Restriction enzymes were then deactivated at 80°C for 20 minutes.  The ligation reaction was assembled by using a molar ratio of plasmid to insert of 1:3. The competent cells were then transformed with the ligation products and plated in LB agar with chloramphenicol. ", ,
PR,Second chance...,26/06/2015,Silvia,"While waiting for the parts to be sequenced we have an alternative: we designed a primer for the proteorhodopsin with the prefix and a strong RBS. With a PCR we easily add the RBS to our gene! The alternative is to perform a standard assembly between the PR with RBS and J61002, which is an unconventional BioBrick with J23100 without RBS and ampicilline resistance.","
",
INTERLAB,We have our devices! ,28/06/2015,Claudio,We made it! Each newly device was confirmed by restriction digestion and by observing the presence of fluorescence on a trans-illuminator. 500 ng of each colony was digested with 1 μl fo EcoRI and SpeI and the expected size of the final plasmids (910 bp) was confirmed by electrophoresis gel. We decided to sequence the 3 devices to make sure the parts assembled correctly once and for all.,,
PR,Digestion,28/06/2015,Silvia,We digested J61002 overnight with SpeI and PstI and the PR with RBS with XbaI and PstI.,,
INTERLAB,Now we need controls,29/06/2015,Elisa,"Positive and negative controls were extracted from the Registry of Standard Parts, transformed into NEB10β cells and amplified. BBa_I20270 (Promoter MeasKit + GFP) is our positive control, while BBa_R0040 (TetR repressible promoter) is our negative control.", ,
PR,Summer of hope,29/06/2015,Silvia,Today is ligation day with our fingers crossed! ,,
BLH,Ready to go,30/06/2015,Veronica,The blh gene was synthetized by a company and today it arrived. Blh is in a pUC57 plasmid and in the next days we'll move it to a pSB1C3 backbone.,,
PR,"Changes are just around the corner
",30/06/2015,Silvia,"We miniprepped the colonies from the ligation (1:2 and 1:4): only the colony A from ligation 1:4 seems to be good. It is absolutely necessary to sequence the part! We have to admit that we are concerned about our gene. We start thinking that maybe during the growth phase of the cells the protein is toxic. While we are waiting for the sequencing results we thought about an alternative, why we don’t use an inducible promoter instead of a costitutive one?",http://2015.igem.org/wiki/images/6/6c/Unitn_pics_notebook_SA.jpg,http://2015.igem.org/wiki/images/6/6c/Unitn_pics_notebook_SA.jpg
PNCB,Re-ligation of mutated pncB in the plasmid with the J23100 promoter ,30/06/2015,Riccardo,It failed again...,,
INTERLAB,Screening positive and negative controls,01/07/2015,Claudio,Positive control was confirmed by restriction digestion with XbaI and SpeI. Negative control was confirmed by Phusion PCR.,,
INTERLAB,What a great day!,02/07/2015,Elisa,We received the results of sequencing… Good news: all the devices are confirmed!, ,
BLH,We have a new device!,02/07/2015,Veronica,"We have the new device with blh gene into a standard plasmid. The next step is to assembly a new device with blh and K274210. This part includes the four genes necessary for the synthesis of B-carotene from FPP, a precursor presents in <i>E. coli</i>.",,
PR,Time to change!,02/07/2015,Silvia,"It is time to change! We choose the araC-pBAD promoter (BBa_K731201), very well characterized by our friends, the 2012 UNITN-Trento iGEM team. It is an arabinose inducible promoter without RBS. We digested overnight the PR with RBS with XbaI and PstI and the araC-pBAD with SpeI and PstI. ",,
INTERLAB,Making our stocks,04/07/2015,Claudio,"After transforming other strains like JM109 and NEB Express with all our parts, we prepared the glycerol stock. Today the -80°C was colonized by more than 400 Eppendorfs, ready to be taken out and measured.",,
INTERLAB,Ready to measure!,06/07/2015,Elisa,"We started measuring the NEB10β. We planned three days of measurements with spectrophotometer and plate reader.
We decided to use a method that takes into consideration biological diversity as well as the reproducibility of the technique. We measured all the devices and their controls.Tecan Infinite 200 Pro plate reader. An aliquot of 150 μl of each sample was placed in a 96-well plate and fluorescence intensities were measured.
Cary Eclipse Varian Spectrofluorimeter: 3mL of each sample was centrifuged and resuspended in 3 ml of PBS, then placed in a cuvette and the fluorescence intensities were measured.",,
BLH,Bad news: SpeI site doesn't work,07/07/2015,Veronica,After two failed assemblies I ran a gel with the plasmid digestion used for the ligation. I saw that the SpeI site of B-carotene didn’t work. Sequencing confirmed this hypothesis.,,
PNCB,New promoters!,08/07/2015,Riccardo,"I tried to insert the mutated pncB in R0010 (Lac promoter) and J23101. Despite the ligation plates were perfect also this experiment was a fail. In this gel we can see J23101 promoter in lane 2 and 3 (about 50 bp), and the lac promoter in lane 5 and 6 (about 240 bp).",http://2015.igem.org/wiki/images/9/94/Unitn_pics_notebook_pncb-j23101%2Bpncb1-2-r0010%2Bpncb1-2.jpg,http://2015.igem.org/wiki/images/8/89/Unitn_pics_notebook_pncb-j23101%2Bpncb1-2-r0010%2Bpncb1-2_thumb.jpg
INTERLAB,"Same measures, different strains",08/07/2015,Elisa,We carried out 3 days of experiments on the NEB Express strain. We used the same parameters as for the NEB10β strain. ,,
BLH,PCR is the solution (maybe),09/07/2015,Veronica,"I tried to regenerate the Spei site through PCR, using the reverse primer. The SpeI site issue is present in both K274200 and K274210 plasmids.",,
PR,Not all bad days are completely bad!,10/07/2015,Silvia,"BAD DAY, SAD DAY! We had bad news from the sequencing: both the 3A and the standard assembly with the constitutive promoter are completely wrong! We our heart in the hands we started miniprepping the ligation of the PR with RBS with araC-pBAD promoter. But not all the bad days are completely lost: form the gel of the colonies we had some good feelings. There is no time to waste: we send the parts to the sequencing without completely loosing our hopes.",http://2015.igem.org/wiki/images/b/b0/Unitn_pics_notebook_Arac.jpg,http://2015.igem.org/wiki/images/b/b0/Unitn_pics_notebook_Arac.jpg
INTERLAB,We are trying new techniques,13/07/2015,Claudio,We performed measurements with FACS (Fluorescence-Activated Cell Sorting) on the NEB10β strain. We also used a Rotor-Gene Q for an in vitro characterization on the transcription production. We produced biological and technical triplicates of every device and control., ,
PR,Good news!,14/07/2015,Silvia,Sequencing confirmed! We are so happy! We have the proteorhodopsin with RBS and the araC-pBAD promoter!,,
INTERLAB,What about measuring another strain?,15/07/2015,Elisa,"We carried out 3 days of measurements on the strain JM109 with the plate reader and the spectrofluorimeter. We are becoming more experienced with this equipment, not to mention the ability to grow different strains at the same time. Cells will no longer have secrets for us!", ,
INTERLAB,We are inundated with data!,20/07/2015,Claudio,For the next few days we have to process the raw data obtained for all the strains with the different techniques. We are very optimistic: data are in line with the expected values and the preliminary analysis look very good!, ,
INTERLAB,Still trying with FACS,24/07/2015,Elisa,We performed measurements with  NEB Express and JM109 strains. We considered biological and technical triplicates for every devices and controls (positive and negative). From a first analysis we can say that different strains with a same device also behave the same.,,
MFC,The prototypes arrived,24/07/2015,Alessandro,Thanks to <b>Martin Hanczyc</b> we have the prototypes with which we can start our tests.,,
MFC,We start the tests,25/07/2015,Alessandro,"We tried with some basic homemade tests, only to understand how the system works (Is it better with stirring or not? Have we correctly set the data logging system?). The system seems to work and we saw a potential of 0,278 V.",,
PNCB,araC is the way!,26/07/2015,Riccardo,"I put pncB* in the plasmid with the promoter araC-pbad. This is th magic gel! At the lane 4 and 10 we have 2 bands, one of the plasmid psb1C3, one araC+pncB*. In the he other lanes we have only the promoter.",http://2015.igem.org/wiki/images/2/25/Unitn_pics_notebook_araC%2BpncB.jpg,http://2015.igem.org/wiki/images/2/25/Unitn_pics_notebook_araC%2BpncB.jpg
MFC,"Tests with the Berkley Strains
",27/07/2015,Alessandro,"Thanks to Dr. Ajo-Franlin from Berkley we had a special strain of <i>E.coli K12</i> and we used it in our <b>mediatorless microbial fuell cell</b>.
In 2 hours of test we reached a maximum potential of 0,238 V.",,
MFC,MFC overnight,28/07/2015,Alessandro,"First all night long test, 16 hours of data logging. Here we tested our strain of <i>E.coli</i> with <b>proteorhodopsin</b> compared to the Berkley strain. Then we added ferricyanide (10mM) to the cathode and methylene blue (235 uM) to the anode. After adding ferricyanide we saw a boost of potential and the system reached a maximum in 0,721 V.",http://2015.igem.org/wiki/images/d/de/Unitn_pics_notebook_grafico444PR.jpg,http://2015.igem.org/wiki/images/d/de/Unitn_pics_notebook_grafico444PR.jpg
MFC,The power of light,01/08/2015,Alessandro,"We started the first experiment with our strain of <i>E.coli</i> in presence and in absence of light.
We induced our bacteria with arabinose (5mM) when the colonies reached an OD of 0.6 and we added retinal (10uM).
The test lasted for 33H. We then added the mediators to see the effect on the system.",http://2015.igem.org/wiki/images/5/5a/Unitn_pics_notebook_accroccomfc.jpg,http://2015.igem.org/wiki/images/3/33/Unitn_pics_notebook_accroccomfc_thumb.jpg
MFC,CONTAMINATION!,03/08/2015,Alessandro,We tested the basal potential of the fresh medium (M9 and LB). We filled the MFC and we started a 16 hour-long test. At the end we discovered that our meida were contaminated!,http://2015.igem.org/wiki/images/0/0e/Unitn_pics_notebook_brodicontaminati.jpg,http://2015.igem.org/wiki/images/0/0e/Unitn_pics_notebook_brodicontaminati.jpg
MFC,The power of contamination,05/08/2015,Alessandro,"We compared the maximum potential of the fresh medium with our <b>pncB</b> <i>E.coli</i> strain in a 16 hour-long test. At the 46' minute we added the mediators to all samples. 
We saw that the contamination in the medium produce a large amount of potential.
",http://2015.igem.org/wiki/images/0/0f/Unitn_pics_notebook_brodiPNCB.jpg,http://2015.igem.org/wiki/images/9/94/Unitn_pics_notebook_brodiPNCB_thumb.jpg
MFC,Sterilized settings,07/08/2015,Alessandro,"We made the same test with fresh medium, but this time we did it under the biological safety cabinet and we took care to sterilize with alchol and UV light all the components of our MFC. We were able to remove the contamination!",http://2015.igem.org/wiki/images/d/df/Unitn_pics_notebook_brodinoncontaminati.jpg,http://2015.igem.org/wiki/images/d/df/Unitn_pics_notebook_brodinoncontaminati.jpg
MFC,Variable resistors,10/08/2015,Alessandro,We tested our PNCB strain compared to a negative control varying the external resistance. We want  to find the max power intensity of our system and generate polarization and power curves.,,
MFC,Berkley power curve,11/08/2015,Alessandro,"After 4 hours of induction with IPTG (1mM), we repeated the previous experiment with the Berkley strain of <i>E.coli K12</i> to generate polarization and power curves. We changed the external resistor every 10 and 20 minutes. We took 10 measures in a range from 10 M&Omega; to 1 &Omega;.",,
INTERLAB,Let's try with the qPCR,13/08/2015,Claudio,"We decided to use the qPCR as our last technique to characterize the NEB10β. It took the whole day to purify the RNA, retrotranscribe it and set the reactions. Hoping to get a good result after all these efforts!",,
INTERLAB,"Yes, yes, yes!",14/08/2015,Claudio,We got the results for the qPCR. Yeah! After analyzing the raw we saw that everything went just as we expected it! We are very satisfied especially because we knew that operating with a reverse transcriptase PCR is not as easy as it may seem.,,